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Expanding the Genetic Code with Unnatural Amino Acids

Senior Seminar. Department of Chemistry. Kevin Cravedi Biochemistry, B.S. Class of 2010. Expanding the Genetic Code with Unnatural Amino Acids. March 11, 2010. Outline. Thesis Background of Method Application of Method Example 1: AzoPhe Example 2: Sulfotyrosine (Sulfohirudin)

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Expanding the Genetic Code with Unnatural Amino Acids

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  1. Senior Seminar Department of Chemistry Kevin Cravedi Biochemistry, B.S. Class of 2010 Expanding the Genetic Code with Unnatural Amino Acids March 11, 2010

  2. Outline Thesis Background of Method Application of Method Example 1: AzoPhe Example 2: Sulfotyrosine (Sulfohirudin) Conclusion

  3. That the incorporation of unnatural amino acids can be expressed in E. Coli with efficiency using the method of Peter G. Schultz, his students and collaborators. Thesis

  4. Background of unnatural amino acid incorporation Incorporation of unnatural amino acid into protein is a pre-translational process.

  5. Reddi, O. S. Recombinant DNA Technology: A Laboratory Manual; Allied Publishers: Mayapuri, New Dehli, 2000; pp 1.

  6. What the novel method looks like. • Wang, L. Wang Lab. Salk Institute for biological studies. http://wang.salk.edu/research.php (accessed Jan 17, 2010).

  7. Application of Method 1 3 2 Pontrello, J. K. 3rd year seminar requirement. http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf (accessed Jan 27, 2010)

  8. 1. Methanoccocus Jannaschii (Mj)

  9. 2. Why use TAG (UAG CODON)

  10. 3. Modification of synthetase to accommodate unnatural amino acid Direct evolution method was implemented in order to rearrange the active site to accomodate the unnatural amino acid. A library of 109 possible synthetase active sites were randomized for one example. 2. Result: Active site specific to unnatural amino acid.

  11. 3. Continuation

  12. Wang, Q.; Parrish, A. R.;Wang, L. Chembiol. 2009, 16(3), 323.

  13. Example (1) Incorporation of Photo-isomerizable Unnatural Amino Acid Phenylalanine-4-azobenzene • Bose, M.; Groff, D.; Xie, J.; Eric, B.; Schultz, P. G. J. Am. Chem. Soc. 2005, 128, 388.

  14. Direct evolution and positive and negative selections were made. Tyr-32, Leu-65, Phe-108, Gln-109, Asp-158, and Leu-162 Tyr32Gly, Leu65Glu, Phe108Ala, Gln109Glu, Asp158Gly, and Leu162His. The active site of AzoPhe synthetase

  15. Example 2: Sulfotyrosine (Tys) (1) building specificity of synthetase and (2) incorporation into protein Tyr32 Leu65 Asp158 Ile159 Leu162 Tyr32Leu, Leu65Pro, Asp158Gly, Ile159Cys, and Leu162Lys Direct evolution and positive and negative selections were made. SDS-PAGE confirms this incorporation. Result: Peak of 7,876 Da only sulfotyrosine. • Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57.

  16. 1) Sulfotyrosine-(maldi-tof) • Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57.

  17. Recombinant hirudin (desulfo-hirudin) is a less potent thrombin inhibitor than native protein as an anticoagulant. This early experiment was done to create natural sulfo-hirudin by incorporation of sulfotyrosine into hirudin. Sulfotyrosine Application

  18. Sulfo-hirudin in THROMBIN COMPLEX • Liu, C. C.; Brustad, E.; Liu, W.; Schultz, P. G. J. Am. Chem. Soc. 2007, 129, 10648.

  19. Placement of unnatural amino acids into proteins with high efficiency has been effectively implemented. conclusion

  20. The end

  21. Dr. Ildliko Kovach Peter G. Schultz and Co. The Chemistry Department Special Thanks

  22. Pontrello, J. K. 3rd year seminar requirement. http://www.chem.wisc.edu/areas/organic/studsemin/pontrello/pontrello-abs.pdf (accessed Jan 27, 2010) • Reddi, O. S. Recombinant DNA Technology: A Laboratory Manual; Allied Publishers: Mayapuri, New Dehli, 2000; pp 1. • Kehoe, J. W.; Bertozzi, C. R. Chem. Biol. 2000, 7, R57. • Bose, M.; Groff, D.; Xie, J.; Eric, B.; Schultz, P. G. J. Am. Chem. Soc. 2005, 128, 388. • Wang, L. Wang Lab. Salk Institute for biological studies. http://wang.salk.edu/research.php (accessed Jan 17, 2010). • Wang, Q.; Parrish, A. R.;Wang, L. Chembiol. 2009, 16(3), 323. • Liu, C. C.; Brustad, E.; Liu, W.; Schultz, P. G. J. Am. Chem. Soc. 2007, 129, 10648. References

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